Project Summary Aminoacyl tRNA synthetases (ARSs) catalyze the formation of aminoacylated tRNA for protein synthesis and other functions with high efficiency and high accuracy. Despite decades of research, our understanding of the reaction mechanism, molecular basis of tRNA specificity, and mechanism of proofreading is incomplete. During the current cycle, we developed rapid kinetics approaches to address these questions, and reported a new functional distinction between class I and class II ARSs, two versions of substrate-assisted catalysis, a new emerging role for RNA in control of both reactions, and new insights into editing. These observations motivated the development of a comprehensive model that describes the entire class II ARS reaction cycle. To fully validate this model, and resolve other longstanding questions in the field, we will (1) correlate the rates of individual binding and chemical steps with structural transitions, employing rapid kinetics and biophysical techniques; (2) Complete the characterization of amino acid editing catalyzed by ThrRS and AlaRS, employing rapid kinetics, resonance energy transfer and other biophysical technique; and (3) characterize small molecule inhibitors of ThrRS, a representative class II ARS, thereby exploiingt our mechanistic work in a directly biomedical context. As yet, a comprehensive kinetic scheme in which all the individual rate constants have been determined has not been reported for any editing ARS. Without such a minimal scheme, one cannot properly assess the relative contributions of pre-transfer, post-transfer and re- sampling mechanisms to overall editing, nor properly calculate the physiological cost of editing. Our studies will result in complete kinetic schemes for representative editing (ThrRS) and non-editing (HisRS) ARSs, and the flip-flop mechanism we have defined for HisRS could be a general feature of dimeric and tetrameric ARSs. In view of recent data implicating editing defects in neurodegenerative diseases, we believe that acquiring this detailed mechanistic information is critical to understanding the linkage between ARS structure/function and the complex nature of eukaryotic protein synthesis, which has a complicated relationship to neural physiology and disease. Our studies will also provide a detailed mechanistic picture of a class II ARS inhibitor, borrelidin, and provide further physiological data useful in identifying borrelidin derivatives that are candidates for anti-angiogenic therapies in the clinic. PUBLIC HEALTH RELEVANCE: Project Narrative Aminoacyl-tRNA synthetases perform an essential function in protein synthesis by attaching amino acids to their transfer RNA adaptors. These enzymes represent validated targets for the development of antibiotics against community acquired infections (i.e., Staphylococcus aureus), and have other, complex roles in brain function that are just beginning to be appreciated. In the future, small molecule regulators directed against these enzymes may provide new avenues to alleviating complex neurological diseases.